Phosphono substituted tetrazole derivatives

ABSTRACT

The present invention relates to the N-phosphonomethyl substituted tetrazole derivatives of formula I ##STR1## and tautomers thereof wherein R 1  is aryl or biaryl; n is zero, 1 or 2 pharmaceutically acceptable mono- or di-ester derivatives thereof in which one or both of the acidic hydroxy groups of the phosphono functional group are esterified in form of a pharmaceutically acceptable mono- or di-ester; and pharmaceutically acceptable salts thereof; pharmaceutical compositions comprising said compounds; methods for preparation of said compounds and for the preparation of intermediates; and methods of treating disorders in mammals which are responsive to the inhibition of neutral endopeptidases by administration of said compounds to mammals in need of such treatment.

SUMMARY OF THE INVENTION

Endogenous atrial natriuretic peptides (ANP), also called atrialnatriuretic factors (ANF) have diuretic, natriuretic and vasorelaxantfunctions in mammals. The natural ANF peptides are metabolicallyinactivated, in particular by a degrading enzyme which has beenrecognized to correspond to the enzyme neutral endopeptidase (NEP) EC3.4. 24.11, also responsible for e.g. the metabolic inactivation ofenkephalins.

The aim of the present invention is to provide novel phosphonosubstituted tetrazole derivatives described below which are useful asneutral endopeptidase (NEP) inhibitors, e.g. as inhibitors of theANF-degrading enzyme in mammals, so as to prolong and potentiate thediuretic, natriuretic and vasodilator properties of ANF in mammals, byinhibiting the degradation thereof to less active metabolites. Thecompounds of the invention are thus particularly useful for thetreatment of conditions and disorders responsive to the inhibition ofneutral endopeptidase EC 3.4. 24.11, particularly cardiovasculardisorders, such as hypertension, renal insufficiency including edema andsalt retention, pulmonary edema and congestive heart failure. By virtueof their inhibition of neutral endopeptidase, the compounds of theinvention may also be useful for the treatment of pain, depression andcertain psychotic conditions. Other potential indications include thetreatment of angina, premenstrual syndrome, Meniere's disease,hyperaldosteronism, hypercalciuria, ascites, glaucoma, asthma andgastrointestinal disorders such as diarrhea, irritable bowel syndromeand gastric hyperacidity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the N-phosphonomethyl substitutedtetrazole derivatives of formula I ##STR2## and tautomers thereofwherein R₁ is aryl or biaryl, preferably monocyclic carbocyclic aryl,monocyclic heterocyclic aryl, or monocyclic carbocyclic aryl which is inturn substituted by monocyclic carbocyclic or monocyclic heterocyclicaryl; n is zero, 1 or 2; pharmaceutically acceptable mono- or di-esterderivatives thereof in which one or both of the acidic hydroxy groups ofthe phosphono functional group are esterified in form of apharmaceutically acceptable mono- or di-ester; and pharmaceuticallyacceptable salts thereof; pharmaceutical compositions comprising saidcompounds; methods for preparation of said compounds and for thepreparation of intermediates; and methods of treating disorders inmammals which are responsive to the inhibition of neutral endopeptidasesby administration of said compounds to mammals in need of suchtreatment.

Compounds of formula I and derivatives thereof, depending on the natureof substituents, possess one or more asymmetric carbon atoms. Theresulting diastereoisomers and optical antipodes are encompassed by theinstant invention.

The tetrazoles can exist in tautomeric forms or mixtures thereof, inwhich the hydrogen can be located on any of the ring nitrogen: such arealso encompassed by the instant invention.

Pharmaceutically acceptable ester derivatives are preferably prodrugderivatives, such being convertible by solvolysis or under physiologicalconditions to the free phosphonic acids of formula I, e.g. thephosphonic acid esters illustrated in European Patent application No.481,214 as prodrugs of phosphonate nucleotide analogs.

Examples of such phosphonic acid esters are aryl, tetrahydronaphthyl andindanyl esters; α-acyloxymethyl esters optionally substituted by loweralkyl, by C₅ -C₇ -cycloalkyl, by aryl or by aryl-lower alkyl; loweralkyl and aryl-lower alkyl esters, each substituted on the α-carbon bycarboxy, esterified or amidated carboxy, or by trichloromethyl.

A preferred embodiment of the invention relates to the compounds offormula II ##STR3## and tautomers thereof wherein R and R' representindependently hydrogen, carbocyclic aryl, 6-tetrahydronaphthyl,5-indanyl, α-(trichloromethyl, carboxyl, esterified carboxyl or amidatedcarboxyl) substituted-(lower alkyl or aryl-lower alkyl), acyloxymethyloptionally monosubstituted on methyl carbon by lower alkyl, by C₅ -C₇-cycloalkyl, by aryl or by aryl-lower alkyl; R₂ represents phenyl, orphenyl substituted by lower alkyl, lower alkoxy, halogen ortrifluoromethyl; or R₂ represents thienyl or furanyl optionallysubstituted by lower alkyl; R₃ and R₄ represent hydrogen, lower alkyl,hydroxy, lower alkoxy or halogen; n is 0, 1 or 2; and pharmaceuticallyacceptable salts thereof.

Preferred are the compounds of formula II and tautomers thereof whereinR and R' independently represent hydrogen, carbocyclic aryl,α-(trichloromethyl, carboxyl, esterified carboxyl or amidated carboxyl)substituted-(lower alkyl or aryl-lower alkyl), (carbocyclic aroyloxy orlower-alkanoyloxy)methyl optionally substituted on the methyl carbon bylower-alkyl, by C₅, C₆ or C₇ -cycloalkyl or by carbocyclic aryl; R₂represents phenyl or phenyl substituted by lower alkyl, lower alkoxy,halogen or trifluoromethyl; R₃ and R₄ represent hydrogen, lower alkyl,lower alkoxy, halogen or trifluoromethyl; and pharmaceuticallyacceptable salts thereof.

Particularly preferred are above said compounds of formula II andtautomers thereof wherein R and R' independently represent hydrogen,lower-alkanoyloxymethyl or lower-alkanoyloxymethyl substituted on methylby lower-alkyl, by cyclohexyl, by cyclopentyl or by phenyl.

Also particularly preferred are said compounds of formula II andtautomers thereof wherein R and R' independently represent hydrogen,5-indanyl, phenyl, or phenyl substituted by one, two or threesubstituents selected from lower alkyl, halogen, lower alkoxy, loweralkanoylamino, trifluoromethyl, lower alkyl-(thio, sulfinyl orsulfonyl), and lower alkoxycarbonyl.

Also particularly preferred are said compounds of formula II wherein Rand R' independently represent hydrogen or α-(carboxy, loweralkoxycarbonyl, carbocyclic arylmethoxycarbonyl, aminocarbonyl or mono-or di-lower alkylaminocarbonyl)-substituted-(lower alkyl or carbocyclicaryl-lower alkyl).

Advantageously, R and R' are either identical, or one of R and R'represents hydrogen while the other of R and R' has any of the othermeanings as defined herein.

A particular embodiment of the invention relates to the compounds offormula IIa ##STR4## and tautomers thereof wherein R and R'independently represent hydrogen, carbocyclic aryl, 5-indanyl,α-(carboxy, lower alkoxycarbonyl, carbocyclic arylmethoxycarbonyl,aminocarbonyl or mono- or di-lower alkylaminocarbonyl)substituted-(lower alkyl or carbocyclic aryl-lower alkyl), or ##STR5##R" represents hydrogen, lower-alkyl, C₅ -, C₆ - or C₇ -cycloalkyl orcarbocyclic aryl; R"' represents lower-alkyl, C₅ -, C₆ - or C₇-cycloalkyl, carbocyclic aryl or carbocyclic aryl-lower alkyl; R₄ and R₄' independently represent hydrogen, lower alkyl, lower alkoxy, halogenor trifluoromethyl; n is zero or 1; and pharmaceutically acceptablesalts thereof.

Preferred are said compounds of formula IIa and tautomers thereofwherein n is zero; R₄ and R₄ ' independently represent hydrogen or loweralkoxy; and other symbols have meaning as defined above; andpharmaceutically acceptable salts thereof.

A preferred embodiment of the invention relates to a compound of formulaIII ##STR6## and tautomers thereof wherein R₄ and R₄ ' representhydrogen or C₁ -C₃ alkoxy; and pharmaceutically acceptable mono- ordi-ester derivatives thereof in which one or both of the acidic hydroxygroups of the phosphono functional group are esterified in form of apharmaceutically acceptable mono- or di-ester; pharmaceuticallyacceptable salts thereof; and optical isomers thereof.

The pharmaceutically acceptable ester derivatives thereof are preferablyprodrug ester derivatives, such being convertible by solvolysis or underphysiological conditions to the free acid of formula III.

Preferred esters are the compounds of formula ##STR7## and tautomersthereof wherein Ar represents phenyl or phenyl substituted by loweralkyl, lower alkoxy, halogen, trifluoromethyl, lower alkanoylamino,lower alkyl-(thio, sulfinyl or sulfonyl) or lower alkoxycarbonyl; or Arrepresents 5-indanyl; R₅ represents hydroxy, lower alkoxy, aryl-loweralkoxy or di-lower alkylamino; R₆ represents hydrogen or lower alkyl; R₄and R₄ ' independently represent hydrogen or C₁ -C₃ alkoxy; andpharmaceutically acceptable salts thereof.

A particular preferred embodiment of the invention relates to abovecompounds wherein n is zero having the (S)-configuration (at theasymmetric carbon bearing the tetrazole ring).

The definitions used herein, unless denoted otherwise, have thefollowing meanings within the scope of the present invention.

Carbocyclic aryl represents preferably monocyclic carbocyclic aryl oroptionally substituted naphthyl.

Monocyclic carbocyclic aryl represents optionally substituted phenyl,being preferably phenyl or phenyl substituted by one to threesubstituents, such being advantageously lower alkyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, cyano, trifluoromethyl, loweralkanoylamino, lower alkyl (thio, sulfinyl or sulfonyl) or loweralkoxycarbonyl.

Optionally substituted naphthyl represents 1- or 2-naphthyl or 1- or2-naphthyl preferably substituted by lower alkyl, lower alkoxy orhalogen.

Heterocyclic aryl represents preferably monocyclic heterocyclic arylsuch as optionally substituted thienyl, furanyl, pyridyl, pyrrolyl orN-lower alkylpyrrolyl.

Optionally substituted furanyl represents 2- or 3-furanyl or 2- or3-furanyl preferably substituted by lower alkyl.

Optionally substituted pyridyl represents 2-, 3- or 4-pyridyl or 2-, 3-or 4-pyridyl preferably substituted by lower alkyl, halogen or cyano.

Optionally substituted thienyl represents 2- or 3-thienyl or 2- or3-thienyl preferably substituted by lower alkyl.

Aryl as in aryl-lower alkyl is preferably phenyl or phenyl substitutedby one or two of lower alkyl, lower alkoxy, hydroxy, lower alkanoyloxy,halogen, trifluoromethyl, cyano, lower alkanoylamino or loweralkoxycarbonyl.

The term "lower" referred to herein in connection with organic radicalsor compounds respectively defines such with up to and including 7,preferably up and including 4 and advantageously one or two carbonatoms. Such may be straight chain or branched.

A lower alkyl group preferably contains 1-4 carbon atoms and representsfor example ethyl, propyl, butyl or advantageously methyl.

A lower alkoxy group preferably contains 1-4 carbon atoms and representsfor example methoxy, propoxy, isopropoxy or advantageously ethoxy.

Aryl-lower alkyl is advantageously benzyl or phenethyl optionallysubstituted by one or two of lower alkyl, lower alkoxy, hydroxy, loweralkanoyloxy, halogen or trifluoromethyl.

The term C₅ -C₇ -cycloalkyl represents a saturated cyclic hydrocarbonradical which preferably contains 5 to 7 ring carbons and is, preferablycyclopentyl or cyclohexyl.

The term cycloalkyl(lower)alkyl represents preferably 1- or2-(cyclopentyl or cyclohexyl)ethyl, 1-, 2- or 3-(cyclopentyl orcyclohexyl)propyl, or 1-, 2-, 3- or 4-(cyclopentyl or cyclohexyl)-butyl.

Esterified carboxy represents preferably lower alkoxycarbonyl, oraryl-lower alkoxycarbonyl.

Amidated carboxy represents preferably aminocarbonyl, mono- or di-loweralkylaminocarbonyl.

Amino-lower alkyl represents preferably amino-(ethyl, propyl or butyl),particularly omega-amino-(ethyl, propyl or butyl).

A di-lower alkylamino group preferably contains 1-4 carbon atoms in eachlower alkyl portion and represents, for example, N,N-dimethylamino,N-methyl-N-ethylamino and advantageously N,N-diethylamino.

A lower alkoxycarbonyl group preferably contains 1 to 4 carbon atoms inthe alkoxy portion and represents, for example, methoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl or advantageously ethoxycarbonyl.

An aryl-lower alkoxycarbonyl group is preferably (monocyclic carbocyclicor heterocyclic)-substituted-lower alkoxy carbonyl, such asbenzyloxycarbonyl.

Lower alkoxycarbonyl-lower alkoxy represents advantageously e.g.1-(ethoxycarbonyl)ethoxy or ethoxycarbonylmethoxy.

Di(lower)alkylamino-lower alkoxy advantageously representsdiethylaminoethoxy.

Halogen (halo) preferably represents fluoro or chloro, but may also bebromo or iodo.

Lower alkanoyl is preferably acetyl, propionyl, butyryl, or pivaloyl.

Lower alkanoyloxy is preferably acetoxy, pivaloyloxy or propionyloxy.

Acylamino represents preferably lower alkanoylamino, aroylamino, oraryl-lower alkoxycarbonylamino such as benzyloxycarbonylamino.

Lower alkanoylamino is preferably acetamido or propionamido.

Aroyl is preferably benzoyl or benzoyl substituted on the benzene ringby lower alkyl, lower alkoxy, halogen or trifluoromethyl.

Acyl represents preferably lower alkanoyl, carbocyclic aryl-loweralkanoyl, aroyl, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl,advantageously lower alkanoyl. Lower alkoxycarbonyl for acyl ispreferably t-butoxycarbonyl (abbreviated t-BOC). Aryl-loweralkoxycarbonyl for acyl is preferably benzyloxycarbonyl (abbreviatedCBZ).

Phosphono derivatized in the form of a pharmaceutically acceptable esterrepresents mono- or di-esters thereof, preferably phosphono derivatizedas mono- or di-prodrug esters such as mono- or di-carbocyclicarylphosphono, e.g. mono- or di-phenylphosphono; mono- ordi-5-indanylphosphono; mono- or di-acyloxymethylphosphono optionallysubstituted on methyl by lower-alkyl, by C₅ -C₇ -cycloalkyl, by aryl(e.g. phenyl) or by aryl-lower alkyl (e.g. benzyl), and wherein acyloxyrepresents lower-alkanoyloxy, C₅ -C₇ -cycloalkanoyloxy, carbocyclicaroyloxy or carbocyclic aryl-lower alkanoyloxy; as mono- or di-(α-loweralkoxycarbonyl-lower alkyl)phosphono; as mono- or di-(α-di-loweralkylaminocarbonyl-lower alkyl)phosphono; also as mono- ordi-(α-trichloromethyl-lower alkyl)phosphono.

Phosphono derivatized as a mono- or di-prodrug ester relates to apharmaceutically acceptable mono- or di-phosphono ester that may beconvertible by solvolysis or under physiological conditions to phosphono(the free phosphonic acid).

Pharmaceutically acceptable salts are pharmaceutically acceptable acidaddition salts for any basic compounds of the invention or salts derivedfrom pharmaceutically acceptable bases for any acidic compounds of theinvention.

Pharmaceutically acceptable salts of basic compounds of the inventionare acid addition salts, which are preferably such of therapeuticallyacceptable inorganic or organic acids, such as strong mineral acids, forexample hydrohalic, e.g. hydrochloric or hydro-bromic acid, sulfuric,phosphoric or nitric acid; aliphatic or aromatic carboxylic or sulfonicacids, e.g. formic, acetic, propionic, succinic, glycollic, lactic,malic, tartaric, gluconic, citric, maleic, fumaric, pyruvic,phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-hydroxybenzoic,salicylic, 4-aminosalicyclic, pamoic, nicotinic, methanesulfonic,ethanesulfonic, hydroxyethanesulfonic, 1,2-ethanedisulfonic acid,benzenesulfonic, p-toluenesulfonic, naphthalenesulfonic, sulfanilic,cyclohexylsulfamic acid, or ascorbic acid.

Pharmaceutically acceptable salts of the acidic compounds of theinvention, e.g. those having a free phosphono hydroxyl group are saltsformed with pharmaceutically acceptable bases, e.g. alkali metal salts(e.g. sodium, potassium salts), alkaline earth metal salts (e.g.magnesium, calcium salts), amine salts (e.g. ethanolamine,diethanolamine, triethanolamine, tromethamine salts).

The novel compounds of the invention are pharmacologically potentneutral endopeptidase enzyme inhibitors which inhibit e.g. thedegradation of atrial natriuretic factors (ANF) in mammals. They thuspotentiate the diuretic and natriuretic effect of exogenous orendogenous ANF in mammals.

The compounds of the invention are thus particularly useful in mammalsas diuretic, natriuretic (saluretic) and antihypertensive agents for thetreatment of e.g. hypertension, congestive heart failure and edema.

As neutral endopeptidase inhibitors, the compounds of the invention alsoinhibit enkephalinase so as to inhibit the degradation of endogenousenkephalins and may thus also be useful for the treatment of pain inmammals.

The above-cited properties are demonstrable in vitro and in vivo tests,using advantageously mammals, e.g. mice, rats, dogs, monkeys or isolatedorgans, tissues and preparations thereof. Said compounds can be appliedin vitro in the form of solutions, e.g. preferably aqueous solutions,and in vivo either enterally, parenterally, advantageouslyintravenously, e.g. as a suspension or in aqueous solution. The dosagein vitro may range between about 10⁻⁵ molar and 10⁻⁹ molarconcentrations. The dosage in vivo may range depending on the route ofadministration, between about 0.01 and 50 mg/kg, advantageously betweenabout 1.0 and 25 mg/kg.

The analgesic activity can be determined by measuring the potentiationof the analgesic effects of enkephalin and derivatives thereof, and byclassical analgesic tests, such as the phenyl-p-benzoquinone inducedwriting test [J. Pharmacol. Exp. Therap. 125, 237 (1959)] and the hotplate test in the mouse [J. Pharmacol. Exp. Therap. 107, 385 (1953).

The antihypertensive activity can be determined e.g. in the DOCA-salthypertensive rat, and/or renal hypertensive rat or dog model.

The diuretic (saluretic) activity can be determined in standard diureticscreens, e.g. as described in "New Antihypertensive Drugs", SpectrumPublications, 1976, pages 307-321, or by measuring the potentiation ofatrial natriuretic factor-induced natriuresis and diuresis in the rat.

The potentiation of ANF can also be determined by measuring the increasein ANF plasma level achieved.

The in vitro inhibition of neutral endopeptidase (NEP) 3.4.24.11 can bedetermined as follows:

The test compound is dissolved in dimethyl sulfoxide or 0.25M sodiumbicarbonate solution, and the solution is diluted with pH 7.4 buffer tothe desired concentration.

Neutral endopeptidase 3.4.24.11 activity is determined by the hydrolysisof the substrate glutaryl-Ala-Ala-Phe-2-naphthylamide (GAAP) using amodified procedure of Orlowski and Wilk (1981). The incubation mixture(total volume 125 μl) contains 4.2 μg of protein (rat kidney cortexmembranes prepared by method of Maeda et al, 1983), 50 mM tris buffer,pH 7.4 at 25° C., 500 μM substrate (final concentration), and leucineaminopeptidase M (2.5 μg). The mixture is incubated for 10 minutes at25° C. and 100 μl of fast garnet (250 μg fast garnet/ml of 10% Tween 20in 1M sodium acetate, pH 4.2) is added. Enzyme activity is measuredspectrophotometrically at 540 nm. One unit of NEP 24.11 activity isdefined as 1 nmol of 2-naphthylamine released per minute at 25° C. at pH7.4. IC₅₀ values are determined, i.e. the concentration of test compoundrequired for 50% inhibition of the release of 2-naphthylamine.

Neutral endopeptidase activity can also be determined using ANF as asubstrate. Atrial natriuretic factor degrading activity is determined bymeasuring the disappearance of rat-ANF (r-ANF) using a 3 minute reversephase-HPLC separation. An aliquot of the enzyme in 50 mM Tris HClbuffer, pH 7.4, is preincubated at 37° C. for 2 minutes and the reactionis initiated by the addition of 4 nmol of r-ANF in a total volume of 50μl. The reaction is terminated after 4 minutes with the addition of 30μl of 0.27% trifluoroacetic acid (TFA). One unit of activity is definedas the hydrolysis of 1 nmol of r-ANF per minute at 37° C. at pH 7.4.IC₅₀ values are determined, i.e. the concentration of test compoundrequired for 50% inhibition of the hydrolysis of ANF.

In vitro testing is most appropriate for the free phosphono acids of theinvention.

Illustrative of the invention,(S)-[2-biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]methylphosphonicacid demonstrates an IC₅₀ of about 1.6 nM in the GAAP in vitro assay.

The effect of the compounds of the invention on rat plasma ANFconcentration can be determined as follows:

Male Sprague-Dawley rats (275-390 g) are anesthetized with ketamine (150mg/kg)/acepromazine (10%) and instrumented with catheters in the femoralartery and vein to obtain blood samples and infuse ANF, respectively.The rats are tethered with a swivel system and are allowed to recoverfor 24 hours before being studied in the conscious, unrestrained state.

In this assay, plasma ANF levels are determined in the presence andabsence of NEP inhibition. On the day of study, all rats are infusedcontinuously with ANF at 450 ng/kg/min. i.v. for the entire 5 hours ofthe experiment. Sixty minutes after beginning the infusion, bloodsamples for baseline ANF measurements are obtained (time 0) and the ratsare then randomly divided into groups treated with the test compound orvehicle. Additional blood samples are taken 30, 60, 120, 180 and 240minutes after administration of the test compound.

Plasma concentrations are determined by a specific radioimmunoassay. Theplasma is diluted (×12.5,×25 and ×50) in buffer containing: 50 mM Tris(pH 6.8), 154 mM NaCl, 0.3% bovine serum albumin, 0.01% EDTA. Onehundred microliters of standards [rANF (99-126)] or samples are added to100 μl of rabbit anti-rANF serum and incubated at 4° C. for 16 hours.Ten thousand cpm of [¹²⁵ I]rANF are then added to the reaction mixturewhich is incubated at 4° C. for an additional 24 hours. Goat anti-rabbitIgG serum coupled to paramagnetic particles is added to the reactionmixture and bound [¹²⁵ I]rANF is pelleted by exposing the mixture to anattracting magnetic rack. The supernatant is decanted and the pelletscounted in a gamma counter. All determinations are performed induplicate. Plasma ANF levels are expressed as a percent of thosemeasured in vehicle-treated animals which received ANF alone (450ng/kg/min i.v.).

Illustrative of the invention,(S)-[2-biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester at a dose of about 10 mg/kg p.o., administered in10% ethanol/polyethylene glycol 400, produces significant increases inplasma ANF levels.

The antihypertensive effect can be determined in desoxycorticosteroneacetate (DOCA)-salt hypertensive rats.

DOCA-salt hypertensive rats (280-380 g) are prepared by the standardmethod. Rats underwent a unilateral nephrectomy and one week later areimplanted with silastic pellets containing 100 mg/kg of DOCA. The ratsare maintained on 1% NaCl/0.2% KCl drinking water for three to fiveweeks until sustained hypertension is established. The antihypertensiveactivity is evaluated at this time.

Two days before an experiment, the rats are anesthetized withmethoxyflurane and instrumented with catheters in the femoral artery tomeasure arterial blood pressure. Forty-eight hours later, baselinearterial pressure and heart rate are recorded during a 1 hour period.The test compound or vehcile is then administered and the samecardiovascular parameters are monitored for an additional 5 hours.

Illustrative of the invention,(S)-[2-biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]methylphosphonicacid diphenyl ester at a dose of 10 mg/Kg p.o., administered in PEG 400,produces a significant reduction in blood pressure in the DOCA-salthypertensive rat model.

The potentiation of the natriuretic effect of ANF can be determined asfollows:

Male Sprague-Dawley rats (280-360 g) are anesthetized with Inactin (100mg/kg i.p.) and instrumented with catheters in the femoral artery,femoral vein and urinary bladder to measure arterial pressure,administer ANF and collect urine, respectively. A continuous infusion ofnormal saline (33 μl/min) is maintained throughout the experiment topromote diuresis and sodium excretion. The experimental protocolconsists of an initial 15 minute collection period (designated aspre-control) followed by three additional collection periods.Immediately after completion of the pre-control period, test compound orvehicle is administered; nothing is done for the next 45 minutes. Then,blood pressure and renal measurements are obtained during a secondcollection period (designated control; 15 min). At the conclusion ofthis period, ANF is administered (1 μg/kg i.v. bolus) to all animals andarterial pressure and renal parameters are determined during twoconsecutive 15 minutes collection periods.

Mean arterial pressure, urine flow and urinary sodium excretion aredetermined for all collection periods. Blood pressure is measured with aGould p50 pressure transducer, urine flow is determined gravimetrically,sodium concentration is measured by flame photometry, and urinary sodiumexcretion is calculated as the product of urine flow and urine sodiumconcentration.

The compounds of the invention are thus particularly useful asinhibitors of neutral endopeptidase, enhancing the potency and durationof action of atrial natriuretic peptide(s). The compounds are thereforeparticularly useful for the treatment of cardiovascular disorders suchas hypertension, edema and salt retention, and cardiac conditions suchas congestive heart failure.

The compounds of the invention can be prepared using processes describedand illustrated below, e.g. by

(a) reacting a compound of the formula ##STR8## wherein R_(A) represents##STR9## wherein R₁ and n have meaning as defined hereinabove, and R_(p)represents a protecting group; with a diester of phosphonic(phosphorous) acid, also named as a disubstituted phosphite of theformula ##STR10## wherein R_(a) and R_(a) ' have meaning as definedherein for R and R', except that R_(a) and R_(a) ' do not representhydrogen, and R_(a) and R_(a) ' may in addition represent lower alkyl oraryl-lower alkyl; and removing the protecting group R_(p) ; or

(b) condensing a protected α-aminotetrazole of the formula ##STR11##wherein R₁ and n have meaning as defined hereinabove and R_(p) is aprotecting group; with a compound of the formula ##STR12## wherein R_(b)and R_(b) ' represent lower alkyl or aryl-lower alkyl, e.g. optionallysubstituted benzyl, and Z represents a leaving group, e.g. a reactiveesterified hydroxyl group, such as trifluoromethylsulfonyloxy; andremoving the protecting group R_(p) ; or

(c) reacting an amide of the formula ##STR13## wherein R₁ and n havemeaning as defined above; R_(b) and R_(b) ' represent lower alkyl oraryl-lower alkyl, e.g. optionally substituted benzyl; and R_(p) andR_(p) ' represent a protecting group;

with a di-lower alkyl azodicarboxylate and a triaryl phosphine (e.g.triphenylphosphine) and with hydrazoic acid (preferably generated insitu) or a reactive azide, such as trimethylsilyl azide, to obtain acompound of the formula ##STR14## wherein R₁, n, R_(b), R_(b) ', R_(p)and R_(p) ' have meaning as defined above; and removing the protectinggroups R_(p) and R_(p) '; or

(d) condensing a compound of the formula ##STR15## wherein R₁, n, R_(p)', R_(b) and R_(b) ' have meaning as defined hereinabove, with hydrazoicacid (preferably generated in situ) or a reactive azide derivative, suchas a trialkyl silyl azide or a trialkyltin azide, and liberating thefree tetrazole from the resulting trialkylsilyl or trialkyltinsubstituted tetrazole by e.g. acid hydrolysis; and removing theprotecting group R_(p) ';

(e) and converting any compound obtained in any said process, in whichany of R_(a), R_(b), R_(a) ' and R_(b) ' represent lower alkyl oraryl-lower alkyl, to a corresponding product of the invention in whichsuch have meaning as defined for R and R' in formula II; and in abovesaid processes, if temporarily protecting any interfering reactivegroup(s), removing said protecting group(s), and then isolating theresulting compound of the invention; and if desired, converting anyresulting compound of the invention into another compound of theinvention; and/or, if desired, converting a free phosphonic acidfunction into a pharmaceutically acceptable ester derivative, orconverting a resulting ester into the free acid or into another esterderivative; and/or, if desired, converting a resulting free compoundinto a salt or a resulting salt into the free compound or into anothersalt, and/or, if desired, separating a mixture of isomers or racematesobtained into the single isomers or racemates, and/or, if desired,resolving a racemate into the optical antipodes.

In starting compounds and intermediates which are converted to thecompounds of the invention in a manner described herein, functionalgroups present, such as phosphonyl, amino and hydroxy groups, areoptionally protected by conventional protecting groups that are commonin preparative organic chemistry. Protected phosphonyl, amino andhydroxy groups are those that can be converted under mild conditionsinto free phosphonyl, carboxyl, amino and hydroxy groups without otherundesired side reactions taking place.

The purpose of introducing protecting groups is to protect thefunctional groups from undesired reactions with reaction components andunder the conditions used for carrying out a desired chemicaltransformation. The need and choice of protecting groups for aparticular reaction is known to those skilled in the art and depends onthe nature of the functional group to be protected (phosphonyl, aminogroup, etc.), the structure and stability of the molecule of which thesubstituent is a part, and the reaction conditions.

Well-known protecting groups that meet these conditions and theirintroduction and removal are described, for example, in J. F. W. McOmie,"Protective Groups in Organic Chemistry", Plenum Press, London, New York1973, T. W. Greene and P. G. M. Wuts, "Protective Groups in OrganicSynthesis", Wiley, N. Y. 1991, and also in "The Peptides", Vol. I,Schroeder and Luebke, Academic Press, London, New York, 1965.

A tetrazole protecting group R_(p) is a group which can be introduced inform of an amide and such is e.g. cyanoethyl, p-nitrophenylethyl, loweralkoxycarbonylethyl, phenylsulfonylethyl and the like. Such tetrazoleprotecting groups can be removed by a retro-Michael deblocking reactionwith a base such as DBN (1,5-diazabicyclo[4.3.0]non-5-ene), an amidine,an alkali metal carbonate or alkoxide, e.g. potassium carbonate,potassium t-butoxide, sodium methoxide in an inert solvent.

The amino protecting group R_(p) ' represents preferably on acylprotecting group such as t-butoxycarbonyl or benzyloxycarbonyl.

A reactive esterified hydroxyl group, such as Z in a compound of theformula VII or XVI, is a hydroxyl group esterified by a strong inorganicor organic acid. Corresponding Z groups are in particular halo, forexample chloro, bromo or preferably iodo, also sulfonyloxy groups, suchas lower alkyl- or arylsulfonyloxy groups, for example (methane-,ethane-, benzene- or toluene-) sulfonyloxy groups, also thetrifluoromethylsulfonyloxy group.

The preparation of compounds of the invention according to process (a),i.e. the condensation of a hexahydrotriazine derivative of formula IVwith a phosphonic acid diester of formula V (the type of reaction isillustrated in U.S. Pat. No. 4,053,505 for the preparation ofN-phosphonomethylglycine) is carried out in an inert solvent such astoluene or benzene, preferably at elevated temperature, to yield e.g. acompound of formula IXa ##STR16## wherein R₁, n, R_(p), R_(a) and R_(a)' have meaning as defined above.

The phosphonic acid (phosphite) diesters of formula V are known or canbe prepared according to methods in the literature, e.g. U.S. Pat. No.3,329,742 for the preparation of diaryl phosphites, by reaction of thealcohol corresponding to R_(a) and R_(a) ' with phosphorus trichlorideas illustrated herein.

Unsymmetrical phosphonic acid diesters can be prepared by first treatinga symmetrical diester, e.g. dibenzyl phosphite, with aqueous base, e.g.aqueous tetramethyl ammonium hydroxide, to obtain a monoester, e.g.monobenzyl phosphite. This can be treated e.g. with an appropriate alkylhalide corresponding to R or R' in formula II, for example anα-alkoxycarbonylalkyl bromide, to obtain a compound of formula V whereinR_(a) is benzyl and R_(a) ' is α-alkoxycarbonylalkyl. Alternatively,monobenzyl phosphite can first be converted to e.g. a mixed anhydride(e.g. with pivaloyl chloride) which is then reacted with an appropriatealcohol or phenol corresponding to R or R' in formula II to obtain acorresponding unsymmetrical diester of formula V. The resultingcondensation product of formula IXa wherein either R_(a) or R_(a) 'represents benzyl can then be converted to a compound of formula IIwherein either R or R' represents hydrogen by selective catalytichydrogenolysis of the benzyl substituent.

As to the hexahydrotriazines of formula IV, such can be prepared fromthe N-acyl amino acid of the formula XI ##STR17## wherein R₁ and n havemeaning as defined herein and acyl represents an easily removable acylprotecting group, as illustrated below.

For example, an N-acylbiarylalanine ester or chain length homologsthereof, e.g. N-t-butoxycarbonyl-4-biphenylalanine methyl ester, isselectively hydrolyzed with dilute base to the correspondingN-acylbiarylalanine, e.g. N-t-butoxycarbonyl-4-biphenylalanine. Thecarboxylic acid is converted e.g. to a mixed anhydride which is thentreated with an amine of the formula XII

    NH.sub.2 --R.sub.p                                         (XII)

wherein R_(p) has meaning as defined herein, and the resulting amide isthen treated under conditions of tetrazole formations, e.g. underconditions described in Tetrahedron Letters 1979, 491 and J. Org. Chem.56 2395 (1991), such as by reaction with an azide such as trimethylsilylazide upon amide activation, with e.g. diethyl azodicarboxylate andtriphenylphosphine, to obtain a protected tetrazole intermediate whichis then N-deacylated to the intermediate of formula VI ##STR18## whereinR₁ and R_(p) have meaning as defined herein.

Condensation of a tetrazole of formula VI, according to the generalknown process for the synthesis of hexahydrotriazine derivatives, e.g.as described in J. Org. Chem. 53, 3113 (1988), with formaldehyde,preferably 37% aqueous formaldehyde, advantageously in a solvent such asa mixture of ethyl acetate and water at room temperature, yields acorresponding hexahydrotriazine derivative of formula IV.

The α-amino acid corresponding to starting materials of formula XI(wherein n is zero) are either known in the art or can be preparedaccording to methods reported in the art. Such can be transformed intothe next higher homologs according to procedures known in the art ormethods described herein to obtain intermediates in which n is 1 or 2.

As to the preparation of the amino acid starting materials in opticallyactive form, such can be prepared e.g. by resolution or by one of thefollowing methods, as illustrated for biphenylalanines:

(1) Adapting a method described in J. Am. Chem. Soc. 1991, 113, 9276 abiarylmethanol, e.g. 4-biphenylylmethanol, is converted to a reactivederivative, e.g. the bromide, which is then condensed with an N-acylderivative of 2,3-diphenyl-6-oxomorpholine, e.g. theN-carbobenzyloxy-(2R,3S)-isomer, in the presence of a strong base suchas sodium bis-trimethylsilylamide, to yield e.g.N-carbobenzyloxy-2(R),3(S),5(S)-6-oxo-2,3-diphenyl-5-(4-biphenylylmethyl)-morpholine. Catalytichydrogenolysis, e.g. using hydrogen and palladium on charcoal ascatalyst, yields the optically active (S)-(+)-4-biphenylalanine.

(2) Alternatively, using the Pd (0)-catalyzed cross-coupling reactiondescribed by W. Shieh et al, J. Organic Chemistry, 57, 379 (1992) thesubstantially optically pure chiral biarylalanines, of the formula##STR19## or the N-acyl and/or carboxy ester derivatives thereof whereinR₂ represents aryl and R₄ have meaning as defined hereinabove, can beprepared by: condensing a reactive esterified optically active tyrosinederivative of the formula ##STR20## wherein the amino and carboxy groupsare in protected form (as N-acyl and esterified carboxy esterderivatives), and Z represents reactive esterified hydroxy(advantageously trifluoromethylsulfonyloxy) with an aryl boronic acid inwhich aryl corresponds to R₂ as defined above, in the presence of apalladium (0) catalyst, in particulartetrakis(triphenylphosphine)palladium (0), and in the presence of ananhydrous base (such as an alkali metal carbonate), in an inert solvent(such as xylene or toluene) at an elevated temperature ranging fromabout 50° to 150° C., and removing any protecting groups as required.

For example, N-t-butoxycarbonyl-tyrosine methyl ester is first convertedto N-t-butoxycarbonyl-4-trifluoromethylsulfonyloxy-phenylalanine methylester (N-t-butoxycarbonyltyrosine triflate methyl ester). This compoundis then condensed with an arylboronic acid (e.g. phenylboronic acid) inthe presence of anhydrous potassium carbonate, and tetrakis(triphenylphosphine) palladium (0) complex as catalyst, in toluenepreferably at an elevated temperature, advantageously at about 100° toobtain N-t-butoxycarbonyl-4-biphenylalanine methyl ester. AfterN-deacylation, substantially optically pure 4-biphenylalanine methylester is obtained with a configuration corresponding to that of thetyrosine derivative used as starting material.

The arylboronic acids are either commercial or can be prepared asdescribed in the literature, e.g. J. Org. Chem. 49, 5237 (1984).

The preparation of the compounds of the invention according to process(b) involves the condensation of a protected tetrazole of formula VI,with a reactive esterified derivative of hydroxymethylphosphonic acid offormula VII, e.g. dimethyl (trifluoromethylsulfonyloxy)methylphosphonate(prepared e.g. according to Organic Synthesis 64, 80 (1985) andTetrahedron Letters 1986, 1477) in a polar solvent, such as methylenechloride, in the presence of a base, e.g. a tertiary amine such asdiisopropylethylamine, at a temperature near room temperature. Theresulting protected tetrazoles can be selectively deprotected to thefree tetrazoles with a base, e.g. DBU in an inert solvent, such asmethylene chloride.

The preparation of the compounds of the invention according to process(c) can be carried out according to methodology for tetrazole ringformation as described under process (a) above for the preparation oftetrazole intermediates of formula VI.

The starting amides of formula VIII can be prepared by condensation ofthe respective carboxylic acids or esters with an amine of formula XII.

The carboxylic acids can in turn be obtained from esters thereof whichcan be prepared according to process (a) above, except that in compoundsrepresented by formula IV, R_(A) is replaced by R_(B) ##STR21## whereinR₁ has meaning as defined above and COR₃ represents esterified carboxyl.

Alternately, the carboxylic acids can be prepared by condensing underreductive amination conditions a compound of the formula XIII ##STR22##wherein R_(b) and R_(b) ' represent lower alkyl or aryl-lower alkyl,with a compound of formula XIV

    R.sub.1 --CH.sub.2 CO--COR.sub.3                           (XIV)

wherein R₁ has meaning as defined hereinabove, and COR₃ representsesterified carboxyl, such as lower alkoxycarbonyl.

The preparation involves the reductive amination of the appropriatepyruvic acid or derivative thereof of formula XIV with a diester ofaminomethylphosphonic acid of formula XIII (e.g. the dimethyl ester), inthe presence of a reducing agent such as hydrogen or sodiumcyanoborohydride under standard reductive amination conditions, e.g. asillustrated in the examples to obtain compounds of formula XV ##STR23##wherein R₁, R_(b), and R_(b) ' have meaning as defined above, and COR₃represents esterified carboxy. The amino protecting group R_(p) ' can beintroduced according to methods well-known in the art.

The aminomethylphosphonic acid diesters of formula XIII are preparedaccording to methods known in the art, for instance by reaction ofphthalimidomethyl bromide with trimethylphosphite [P(OCH₃)₃ ] to obtainthe corresponding dimethyl phthalamidomethyl phosphonate which isconverted with hydrazine to dimethyl aminomethylphosphonate.

As to the pyruvic acid esters of formula XIV, such are known in the artor are in turn prepared by methods analogous to those used for thepreparation of substituted pyruvic acids, e.g. by condensation of e.g.the methyl ester of a biarylacetic acid with diethyl oxalate in thepresence of a base, e.g. potassium t-butoxide, followed by hydrolyticdecarboxylation.

The preparation of the compounds of the invention according to process(d) can be carried out according to procedures known in the art for thepreparation of tetrazoles from nitriles e.g. as described in J. Am.Chem. Soc. 80, 3908 (1958) and J. Org. Chem. 56, 2395 (1991).

Hydrazoic acid is preferably generated from ammonium chloride/sodiumazide in situ.

The starting nitriles can be prepared in a conventional manner from thecorresponding primary amides which can in turn be obtained from thecarboxylic acid esters, described under process (c), by treatment withammonia.

If a trialkylsilyl azide (such trimethylsilyl azide) or a trialkyltinazide is used, the resulting tetrazole may be substituted by trialkyltinor trialkylsilyl. Such groups may be removed by hydrolysis, e.g. diluteacid.

The conversion according to process (e) of products obtained in theabove processes, e.g. of formula IXa and IXb wherein R_(a), R_(a) ',R_(b) and R_(b) ', represent lower alkyl or aryl-lower alkyl tocompounds of formula I can be carried out using known reagents forconverting phosphonic acid esters to phosphonic acids, e.g. hydrobromicacid in glacial acetic acid, trimethylsilyl bromide, or by catalytichydrogenation when such represent optionally substituted benzyl.

The compounds of the invention so obtained, can be converted into eachother according to conventional methods. Thus, any resulting free acidcan be converted into a corresponding metal, ammonium or acid additionsalt respectively, by reacting it with an equivalent amount of thecorresponding base, e.g. said free acids with alkali or ammoniumhydroxides or carbonates. Any resulting salt may also be converted intothe free compound, by liberating the latter with stronger acids. In viewof the close relationship between the free compounds and the saltsthereof, whenever a compound of the invention, or intermediate, isreferred to in this context, a corresponding salt is also intended,provided such is possible or appropriate under the circumstances.

The compounds, including their salts, may also be obtained in the formof their hydrates, or include other solvents used for thecrystallization.

Furthermore, the functional derivatives of the free acids of formula I,wherein the phosphono hydroxyl groups are esterified by identical ordifferent radicals may be prepared by condensing a free phosphonic acidof formula I or a mono-ester derivative thereof with an esterifyingagent of the formula XVI

    R.sub.7 --Z                                                (XVI)

wherein Z represents hydroxy or a reactive esterified hydroxyl group;and R₇ represents an esterifying radical as defined herein for thephosphonyl esters (e.g. R and R').

The esterification of the phosphonyl group, with a compound of formulaXVI wherein Z represents a reactive esterified hydroxyl group, isperformed in a manner known per se, in the presence of for example anorganic base, such as an organic amine, for example a tertiary amine,such as tri-lower alkylamine, for example trimethylamine, triethylamineor ethyl-di-isopropylamine, an N,N-di-lower-alkyl-aniline, for exampleN,N-di-methylaniline, or a quaternary ammonium base, such as atetraalkylammonium hydroxide, carbonate or hydrogen carbonate, forexample in which alkyl is e.g. methyl, ethyl, propyl, isopropyl, butyl,or the like, or an alkali metal salt of bis-trialkylsilylamide (e.g.trimethyl) optionally in the presence of a crown ether such as18-crown-6 in a suitable inert solvent or solvent mixture, e.g.acetonitrile, toluene, and the like.

The compounds of formula XVI are known or can be prepared by methodswell-known to the art.

A compound of the formula XVI wherein Z is a reactive esterifiedhydroxyl group can be prepared in situ. For example, a compound of theformula XVI wherein Z is chloro can be converted by treatment withsodium iodide in a solvent, for example in acetone or acetonitrile, intoa compound of the formula XVI wherein Z is iodo; or esterification canbe carried out with a chloro compound of the formula XVI in the presenceof sodium iodide.

Esters of the invention (phosphonic acid di-esters), can be converted tocompounds of the invention with one or two free phosphonyl hydroxygroups using methods and conditions generally known in the art andillustrated herein. Depending on type of ester involved, useful reagentsinclude aqueous acids or bases; also anhydrous reagents such astrialkylsilyl halides, hydrobromic acid in glacial acetic acid; alsohydrogen and a hydrogenolysis catalyst. For instance, dialkyl esters canbe converted to the free phosphonic acids by treatment with hydrobromicacid in glacial acetic acid, e.g. at room temperature or elevatedtemperature.

Any benzyl esters can be selectively hydrogenolyzed with e.g. hydrogenin the presence of a catalyst such as palladium on charcoal.

Phosphono diesters wherein the esterifying groups (R and R') representα-acyloxyalkyl can be converted to corresponding phosphono monoesters(wherein one of R and R' represents hydrogen) by treatment with onemolar equivalent of an aqueous base, e.g. 1N sodium hydroxide.

Phosphono diesters wherein the esterifying groups (e.g. R and R' informula II) represent aryl (for instance the compounds of formula IIIa)can advantageously be converted to the corresponding phosphonomonoesters (wherein one of R and R' represents hydrogen) using diluteaqueous acid (e.g. dilute hydrochloric acid) in a polar water misciblesolvent such as acetonitrile.

Furthermore, phosphono diesters wherein the esterifying groups representaryl can first be converted to the corresponding phosphono diesterswherein the esterifying groups represent e.g. methyl, by treatment withmethanol in the presence of potassium fluoride and a crown ether such as18-crown-6. Subsequent treatment with hydrobromic acid in glacial aceticacid yields the free phosphonic acid.

In the case mixtures of stereoisomers or optical isomers of the abovecompounds are obtained, these can be separated into the single isomersby methods in themselves known, e.g., by fractional distillation,crystallization and/or chromatography and racemic products can beresolved into the optical antipodes.

The above-mentioned reactions are carried out according to standardmethods, in the presence or absence of diluents, preferably such as areinert to the reagents and are solvents thereof, of catalysts, alkalineor acidic condensing or said other agents respectively and/or inertatmospheres, at low temperatures, room temperature or elevatedtemperatures, preferably near the boiling point of the solvents used, atatmospheric or superatmospheric pressure.

The invention further includes any variant of said processes, in whichan intermediate product obtainable at any stage of the process is usedas a starting material and any remaining steps are carried out, or theprocess is discontinued at any stage thereof, or in which the startingmaterials are formed under the reaction conditions, or in which thereaction components are used in the form of their salts or opticallypure antipodes. Mainly those starting materials should be used in saidreactions, that lead to the formation of those compounds indicated aboveas being preferred.

The present invention additionally relates to the use in mammals of thecompounds of the invention and their pharmaceutically acceptable,non-toxic acid addition salts, or pharmaceutical compositions thereof,as medicaments, e.g. as neutral endopeptidase inhibitors, e.g. for thetreatment of cardiovascular disorders such as hypertension, edema, saltretention and congestive heart failure.

The present invention also relates to the use of the compounds of theinvention for the preparation of pharmaceutical compositions especiallypharmaceutical compositions having neutral endopeptidase inhibitingactivity, and e.g. antihypertensive or saluretic activity.

The pharmaceutical compositions according to the invention are thosesuitable for enteral, such as oral or rectal, transdermal and parenteraladministration to mammals, including man, for the treatment ofcardiovascular disorders, such as hypertension, comprising an effectiveamount of a pharmacologically active compound of the invention or apharmaceutically acceptable salt thereof, alone or in combination withone or more pharmaceutically acceptable carriers.

The pharmacologically active compounds of the invention are useful inthe manufacture of pharmaceutical compositions comprising an effectiveamount thereof in conjunction or admixture with excipients or carrierssuitable for either enteral or parenteral application. Preferred aretablets and gelatin capsules comprising the active ingredient togetherwith a) diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) lubricants, e.g. silica, talcum, stearicacid, its magnesium or calcium salts and/or polyethyleneglycol; fortablets also c) binders, e.g. magnesium aluminum silicate, starch paste,gelatin, tragacanth, methylcellulose, sodium carboxymethylcelluloseand/or polyvinylpyrrolidone; if desired, d) disintegrants, e.g.starches, agar, alginic acid or its sodium salt, or effervescentmixtures; and/or e) absorbents, colorants, flavors and sweeteners.Injectable compositions are preferably aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, the compositions may also containother therapeutically valuable substances. Said compositions areprepared according to conventional mixing, granulating or coatingmethods, respectively, and contain about 0.1 to 75%, preferably about 1to 50%, of the active ingredient.

Suitable formulations for transdermal application include an effectiveamount of a compound of the invention with carrier. Advantageouscarriers include absorbable pharmacologically acceptable solvents toassist passage through the skin of the host. Characteristically,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound, optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin.

A unit dosage for a mammal of about 50 to 70 kg may contain betweenabout 5 and 100 mg of the active ingredient. The dosage of activecompound is dependent on the species of warm-blooded animal (mammal),the body weight, age and individual condition, and on the form ofadministration.

The following examples are intended to illustrate the invention and arenot to be construed as being limitations thereon. Temperatures are givenin degrees Centigrade. If not mentioned otherwise, all evaporations areperformed under reduced pressure, preferably between about 15 and 100 mmHg. Optical rotations are measured at room temperature at 589 nm (D lineof sodium).

The prefixes R and S are used to indicate the absolute configuration ateach asymmetric center and the corresponding enantiomers.

The tetrazole derivatives are named as 1-H or 1-substituted compounds.However, such may exist as tautomeric 2-H or 2-substituted compounds oras a mixture of said tautomeric forms.

EXAMPLE 1

To a stirred solution of(S)-[2-(biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid dimethyl ester (0.41 g, 1.06 mmol) in acetic acid (3.5 mL) heatedto 90° is added 9N HCl (15 mL). Heating is continued for 12.5 hours,then the reaction mixture is stirred at room temperature for 12 hours.The excess of HCl is removed under reduced pressure, then water isadded. The solid is filtered off and washed with water, then ether. Thesolid is dried at 70° under high vacuum for 4 hours then at roomtemperature for 18 hours.(S)-[2-(Biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid is obtained as a colorless powder, m.p.: 272° (dec), [α]_(D)=+58.4° (c 0.9, 0.1N NaOH).

The starting material is prepared as follows:

(S)-2-t-Butoxycarbonylamino-3-biphenyl-4-yl-propionic acid (J. Org.Chem., 1992, 57, 379; 20 g, 58 mmol) in ethyl acetate (300 mL) andmethylene chloride (75 mL) is cooled under nitrogen to 0° with an icebath and treated with N-methylmorpholine (6.5 mL, 58 mmol). Isobutylchloroformate (7.6 mL, 58 mmol) is added dropwise. After 5 minutes ofstirring, 3-aminopropionitrile (4.52 g, 64 mmol) in methylene chloride(50 mL) is added over 4 minutes. Stirring is continued for 1 hour at 0°then for 4 hours at room temperature. Ethyl acetate (300 mL) is addedand the solution is successively washed with cold water (100 mL),saturated sodium bicarbonate (100 mL) and water (100 mL). The organiclayer is dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure. The residue is triturated with ether (150 mL).The solid is filtered off, washed with cold ether and dried under highvacuum to give(S)-2-t-butoxycarbonylamino-3-biphenyl-4-yl-N-(2-cyanoethyl)-propionamideas a colorless crystalline solid, m.p. 163°-164°; [α]_(D) =+3.58° (c 1,CHCl₃).

To a stirred solution of(S)-2-t-butoxycarbonylamino-3-biphenyl-4-yl-N-(2-cyanoethyl)-propionamide(18 g, 45.7 mmol) in THF (375 mL) under nitrogen is addedtriphenylphosphine (12 g, 45.7 mmol). The solution is cooled to 0° andtreated with diethyl azodicarboxylate (DEAD, 7.2 mL, 24.3 mmol) followedby trimethylsilyl azide (3.2 mL, 24.3 mmol). The solution is warmed upto room temperature and stirred for 18 hours. One additional equivalentof triphenyl phosphine (12 g, 45.7 mmol), DEAD (7.2 mL, 24.3 mmol) andtrimethylsilyl azide (3.2 mL, 24.3 mmol) are added and stirring iscontinued for 3 days. The solution is cooled to 0°. Ceric ammoniumnitrate (2.2 L; 10% aqueous solution) is added dropwise. After 20minutes of stirring, extraction is carried out with methylene chloride(3×200 mL). The combined organic layers are dried over anhydrous sodiumsulfate, then filtered and concentrated in vacuo. The residue is takenup in ethyl acetate (500 mL) and hexane (50 mL). A small amount of(S)-3-[5-(1-t-butoxycarbonylamino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitrilecrystallizes out. The filtrate is concentrated and the residue purifiedby flash-chromatography eluting with a gradient of ethyl acetate inhexane (30% to 50%). The pure fractions of product (Rf=0.6 in 1/1 ethylacetate/hexane) are combined and concentrated under reduced pressure togive(S)-3-[5-(1-t-butoxycarbonylamino-2-biphenyl-4-yl-ethyl)tetrazol-1-yl]-propionitrileas a colorless crystalline solid, m.p. 184°-185°, [α]_(D) =-2.90° (c0.89, CHCl₃).

(S)-3-[5-(1-t-butoxycarbonylamino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitrilecan also be prepared as follows:

To a stirred solution of(S)-2-t-butoxycarbonylamino-3-biphenyl-4-yl-N-(2-cyanoethyl)-propionamide(20 g, 51.2 mmol) in acetonitrile (220 mL) under nitrogen is addedtriphenyl phosphine (33.6 g, 128 mmol). The suspension is cooled to 0°.Diisopropyl azodicarboxylate (24.8 mL, 125.6 mmol) is placed in anaddition funnel. In a separate addition funnel is placed trimethylsilylazide (16.8 mL, 127.2 mmol). The two reagents are introduced dropwise,allowing the azoester to be added about 1 minute faster than the silylazide and keeping the reaction temperature below 10°. The slightlyyellow suspension is gradually (10°/hour) warmed up to 35°. The reactionis then monitored by TLC (ethyl acetate/hexane:3/1) until practicallycomplete conversion is observed. A clear solution is then usuallyobtained. The excess of azide is destroyed by cooling the solution to10° and adding sodium nitrite (3.2 g) in water (16 mL), followed byacetic acid (16 mL). The mixture is stirred at 20°-25° for at least 2hours, until a nitrite-free aliquot of the acetonitrile layer shows anegative zide test (ferric chloride paper). The lower salt phase isseparated and the organic layer is concentrated in vacuo at 45°. Thesemi-solid residue is dissolved in isopropanol at 80°. Some insolublematerial is filtered from the hot solution. The title compoundcrystallizes at about 60° after seeding. The suspension is cooled to 25°within 2 hours then left at 0°-5° for 1 hour. The product is filtered,washed with cold ispropanol (3×20 mL) and dried under reduced pressureat 50° to constant weight to obtain(S)-3-[5-(1-t-butoxycarbonylamino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitrile.

(S)-3-[5-(1-t-butoxycarbonylamino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitrile(9.22 g, 22 mmol) is dissolved in methylene chloride (135 mL) undernitrogen. To the stirred solution is added trifluoroacetic acid (50 mL).After 50 minutes, the solution is concentrated under reduced pressureand the residue is treated with ether (200 mL). The amorphous solid isfiltered off, washed with ether and dried under vacuum at 45° for 2hours, then at room temperature for 18 hours to yield(S)-3-[5-(1-amino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitriletrifluoroacetate salt, m.p. 211°-212°, [α]_(D) =+23.35° (c 1.05, DMSO).

(S)-3-[5-(1-amino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitriletrifluoroacetate salt (1.0 g, 2.3 mmol) is suspended in saturated sodiumbicarbonate (10 mL) and extracted in methylene chloride (2×25 mL). Theorganic layer is dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo to give the corresponding free base as anamorphous solid (0.73 g, 100%). The latter is dissolved in methylenechloride (8 mL) and treated with diisopropylethyl-amine (0.57 mL, 3.29mmol). To the solution at 0° is added dimethylphosphonomethyltrifluoromethylsulfonate (Tetrahedron Lett., 1986, 1477) (0.81 g, 2.99mmol) and the reaction mixture is stirred for 75 minutes. After warmingto room temperature, stirring is continued for 24 hours. Ethyl acetate(60 mL) is added and the solution is washed successively with cold 1NHCl (20 ml), water, cold sodium bicarbonate solution and water. Theorganic layer is dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. The residue is purified by flash-chromatographyusing a gradient of methanol (0 to 1%) in ethyl acetate as eluent.(S)-[1-[1-(2-Cyanoethyl)-1H-tetrazol-5-yl]-2-(biphenyl-4-yl)ethylamino]-methylphosphonicacid dimethyl ester is obtained as a colorless oil, [α]_(D) =+3.76° (c0.77, CHCl₃).

To a solution of(S)-[1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-2-(biphenyl-4-yl)-ethylamino]-methylphosphonicacid dimethyl ester (580 mg, 1.32 mmol) in THF (6 mL) and methanol (6mL) is added dropwise 1N sodium hydroxide (1.52 mL, 1.52 mmol). After 2hours, the reaction mixture is treated with 1N hydrochloric acid (1.8mL, 1.8 mmol). The organic solvents are removed by concentration underreduced pressure and the residue is treated with water (5 mL) andextracted with methylene chloride (3×15 mL). The combined organic layersare dried over anhydrous sodium sulfate, filtered and concentrated invacuo.(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid dimethyl ester is crystallized from ether, m.p.: 117°-118°, [α]_(D)=-34.67° (c 0.8, CHCl₃).

EXAMPLE 2

Similarly to the procedures in example 1, the following are prepared:

1)(S)-[2-(2-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid; m.p.: 239°-241° (dec.), [α]_(D) =25.81° (c 0.61, NaOH 1N), beingthe compound of formula III wherein R₄ is 2-methoxy.

2)(S)-[2-(2'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid; m.p.: 250° (dec.), being the compound of formula III wherein R₄ 'is 2'-methoxy.

The starting protected amino acid,(S)-2-t-butoxycarbonylamino-3-(2'-methoxy-biphenyl-4-yl)-propionic acid,is prepared as follows:

To a cold (-78°) solution of n-butyllithium (2.5 M in hexane, 5.1 mL,12.8 mmol) in dry THF (20 mL) under nitrogen is added dropwise2-bromoanisole (1.3 mL, 10.7 mmol). The mixture is stirred for 45minutes, then treated with trimethylborate (3.64 mL, 32 mmol). Thesolution is allowed to warm up to room temperature and stirring iscontinued for 18 hours. A 0.5% HCl solution is added to reach pH 6.5.The product is extracted in methylene chloride (2×40 mL). The organiclayer is dried over anhydrous sodium sulfate, filtered and concentratedin vacuo. The residue is triturated with hexane to give the boronic acidas a white solid (m.p. 102°). Palladium-catalyzed coupling to (S)-N-tBOCtyrosine O-trifluoromethanesulfonate methyl ester is carried outaccording to J. Org. Chem., 1992, 57, 379 to give(S)-2-t-butoxycarbonylamino-3-(2'-methoxy-biphenyl-4-yl)-propionic acidmethyl ester.

A solution of(S)-2-t-butoxycarbonyl-amino-3-(2'-methoxybiphenyl-4-yl)-propionic acidmethyl ester (4.1 g in methanolic 1N sodium hydroxide (60 mL) is stirredat room temperature for 3 hours. Ether (30 mL) and water (30 mL) areadded. The aqueous layer is separated and acidified with concentratedHCl, then extracted with ether (2×20 mL) and dried over anhydrous sodiumsulfate. Evaporation of the solvent under reduced pressure gives(S)-2-t-butoxycarbonyl-amino-3-(2'-methoxybiphenyl-4-yl)-propionic acid.

3)(S)-[2-(3-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid;

4)(S)-[2-(3'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid.

EXAMPLE 3

To a solution of(S)-[1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-2-(biphenyl-4-yl)ethylamino-methylphosphonicacid diphenyl ester (0.5 g; 0.89 mmol) in methylene chloride (10 mL)under nitrogen is added 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU, 0.66mL; 4.4 mmol). After 3 hours, the solution is diluted with ethyl acetate(60 mL), washed with ice cold HCl 1N (15 mL) and water (2×15 mL). Theorganic phase is dried over magnesium sulfate and filtered through aplug of silica gel to remove traces of polar impurities. The silica gelis washed with ethyl acetate (125 mL), then with methylenechloride/methanol (95/5) (20 mL). The solvents are removed under reducedpressure to give a glassy residue which is redissolved in warm ethylacetate (5 mL) and treated with hexane (5 mL). Crystallization occursupon cooling to 0° for 45 minutes. The solid is filtered off, washedwith ethyl acetate/hexane (1/1 ) and dried under high vacuum for 1 hourat 45° and 20 hours at room temperature.(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester is obtained as a colorless crystalline solid, m.p.:128°-129°, [α]_(D) =-39.5° (c 0.86, CHCl₃).

The starting material is prepared as follows:

(S)-3-[5-(1-amino-2-biphenyl-4-yl-ethyl)-tetrazol-1-yl]-propionitriletrifluoroacetate salt (2.5 g, 5.7 mmol) is suspended in ice coldsaturated sodium bicarbonate (50 mL) and the free base is extracted withmethylene chloride (3×25 mL). The organic layer is dried over magnesiumsulfate, filtered and concentrated in vacuo. To the residue (free base)dissolved in ethyl acetate (25 mL) at 0° is added an aqueous solution offormaldehyde (0.6 mL; 37% in water; 8 mmol). The reaction mixture iswarmed up to room temperature over 2 hours and stirred for 14 hours.Cold water is added (40 mL) and the organic layer is dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure to give the hexahydrotriazine as a colorless amorphous solidwhich is dried under high vacuum. To the crude hexahydrotriazine (1.7 g;5.13 mmol) in toluene (25 mL) and THF (10 mL) under nitrogen is addeddiphenyl phosphite (1.5 mL; 6.15 mmol). The mixture is heated to 70° for90 minutes then at room temperature for 18 hours. THF is evaporated invacuo and the residue taken up in ethyl acetate (30 mL), washed withcold 0.5N HCl (15 mL), water (15 mL) and brine (15 mL). The organiclayer is dried over sodium sulfate, filtered and concentrated underreduced pressure. The oily residue is dissolved in warm (40°-45°) ethylacetate. Hexane (30 mL) is added. The product crystallizes slowly, andthe mixture is cooled to 0° for 18 hours. The solid is filtered off andwashed with ethyl acetate/hexane (1/1) before being dried under highvacuum at 45°.(S)-[1-[1-(2-Cyanoethyl)-1H-tetrazol-5-yl]-2-(biphenyl-4-yl)-ethylamino]-methylphosphonicacid diphenyl ester is obtained as a colorless crystalline solid, m.p.:133°-134°, [α]_(D) =-12.59° (c 0.94, CHCl₃).

EXAMPLE 4

Similarly prepared according to the procedures described in example 3are:

1)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-3-methylphenyl ester;

The starting di-(3-methylphenyl)phosphite is prepared as follows:

To a stirred solution of m-cresol (5.1 g, 47 mmol) in methylene chloride(5 mL) cooled to 0° under nitrogen is aded methanol (0.97 mL, 24 mmol).Phosphorus trichloride (2 mL, 23 mmol) is then added dropwise over 20minutes. The flask is vented to allow the hydrochloric acid formed toexcape, then the mixture is allowed to warm up slowly to roomtemperature and stirred to 10 hours. The solvent is removed underreduced pressure and the residue is dried under high vacuum to yielddi-(3-methylphenyl)phosphite.

Other diaryl phosphites used as starting materials for compounds listedbelow are similarly prepared from the corresponding substituted phenol.

2)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-3,5-dimethylphenyl ester, m.p.: 64°-67°, [α]_(D) =-32.71° (c0.75, CHCl₃);

3)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-3-methoxyphenyl ester;

4)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-4-acetamidophenyl ester;

5)(S)-[2-(2-methoxybiphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester;

6)(S)-[2-(2'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester;

7)(S)-[2-(3-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester;

8)(S)-[2-(3'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester.

EXAMPLE 5

To a stirred solution of(S)-[2-biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid diphenyl ester (0.36 g, 0.7 mmol) in THF (7 mL) is added 2N HCl(3.5 mL). After stirring at room temperature for 18 hours, theprecipitate is filtered off, washed with THF/water (1/1) (5 mL) thenwater (10 mL). The solid is stirred in ethyl acetate (3 mL) for 1.5hours, filtered and dried under vacuum.(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid monophenyl ester is obtained as a colorless solid, m.p.: 262°(dec), [α]_(D) =+9.76° (c, 0.76, TFA).

EXAMPLE 6

Similarly prepared according to the procedure described in example 5are:

1)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid mono-3,5-dimethylphenyl ester,

2)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid mono-3-methylphenyl ester,

3)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid mono-3-methoxyphenyl ester,

4)(S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid mono-4-acetamidophenyl ester,

5)(S)-[2-(2-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid monophenyl ester,

6)(S)-[2-(2'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid monophenyl ester,

7)(S)-[2-(3-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid monophenyl ester,

8)(S)-[2-(3'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid monophenyl ester.

EXAMPLE 7

(S)-[2-Biphenyl-4-yl-1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester (2.96 g, 5.06 mmol) is dissolved inmethylene chloride (35 mL) under nitrogen and treated with DBU (3.8 mL,25.3 mmol). The resulting pale yellow solution is stirred at roomtemperature for 3.5 hours. The reaction mixture is added to a wellstirred mixture of ice and 0.5M sodium dihydrogenophosphate (75 mL). Theorganic phase is separated. The aqueous layer is extracted withmethylene chloride (2×25 mL). The combined organic layers are dried overmagnesium sulfate and filtered through a plug of silica gel. Afterwashing the silica gel with 3% of methanol in methylene chloride, thefiltrate is concentrated in vacuo and the residue purified byflash-chromatography, eluting with 4% of methanol in methylene chloride.(S)-[2-Biphenyl-4-yl-1-[1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester is recrystallized from ethylacetate-hexane to give a colorless crystalline solid; m.p.: 123°-124°,[α] _(D) =-43.68° (c 0.76, CHCl₃).

The starting material is prepared as follows:

To a stirred solution of ethyl glycolate (7.2 g, 69 mmol) in cold (icebath) methylene chloride (8 mL) is added dropwise phosphorus trichloride(2 mL, 23 mmol). The solution is stirred at room temperature for 16hours. The solution is concentrated under high vacuum and the obtainedcrude di-(ethoxycarbonylmethyl)phosphite is used directly.

Alternately, di-(ethoxycarbonylmethyl)phosphite can be prepared asfollows:

To a stirred solution of dry phosphorous acid (1 g, 12.2 mmol) inanhydrous acetonitrile (10 mL) under nitrogen, is added at 0°diisopropyl ethylamine (4.25 mL) followed by ethyl bromoacetate (2.72mL, 24.4 mmol). The mixture is allowed to warm slowly to roomtemperature and stirred for 18 hours. The solvent is removed underreduced pressure and the residue taken in ethyl acetate. The solidmaterial is filtered off and the filtrate is washed successively withcold 1N hydrochloric acid and water. The organic layer is dried overanhydrous magnesium sulfate, filtered and concentrated in vacuo. Theresidue is dried under high vacuum, yielding the title product as anamber oil characterized by H¹ -NMR in CDCl₃ (P-H:7.23 ppm, J=625 Hz).

A solution of the hexahydro-triazine obtained in example 3 (3.2 g, 9.66mmol) in toluene (30 mL) is treated with di-(ethoxycarbonylmethyl)phosphite (3.2 g, 12.6 mmol) and the solution is heated under nitrogento 70° for 1.5 hours, then stirred at room temperature for 20 hours. Thesolution is diluted with ethyl acetate (30 mL), washed successively withcold water, cold HCl 0.5N (30 mL), cold water (2×25 mL) and dried overanhydrous sodium sulfate. The filtrate is concentrated and the residuepurified by flash-chromatography, eluting with a gradient of ethylacetate in hexane (70% to 75%).(S)-[2-Biphenyl-4-yl-1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester is obtained as a colorless oil.

EXAMPLE 8

Similarly prepared according to the procedures described in example 7are:

1)(S)-[2-biphenyl-4-yl-1-[1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(isopropyloxycarbonylmethyl) ester, oil;

2)(S)-[2-biphenyl-4-yl-1-[1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(N,N-dimethyl-carbamoylmethyl) ester, m.p. 115°-119°;

3)(S)-[2-biphenyl-4-yl-1-[1H-tetrazol-5-yl]-ethylamino]-methylphosphonicacid di-(2-trichloroethyl) ester;

4)(S)-[2-(2-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester;

5)(S)-[2-(2'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester;

6)(S)-[2-(3-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester;

7)(S)-[2-(3'-methoxy-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid di-(ethoxycarbonylmethyl) ester.

EXAMPLE 9

(a) To a stirred solution of(R)-[1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-2-(biphenyl-4-yl)-ethylamino]-methylphosphoicacid diphenyl ester (0.35 g, 0.62 mmol) in methanol (6 mL) undernitrogen, is added potassium fluoride (0.36 g, 6.2 mmol) and 18-crown-6(25 mg, 0.09 mmol). The solution is warmed in a pre-heated oil bath at80° and refluxed gently for 10 minutes and then cooled to roomtemperature. 1N Hydrochloric acid (5 mL) is added and the methanol isremoved under reduced pressure. The residue is treated with water (10mL) and extracted with methylene chloride (3×10 mL). The combinedorganic layers are dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo. The residue is dissolved in a 1/1 mixture ofmethanol and THF (6 mL) and treated with 1N sodium hydroxide (1.06 mL,1.06 mmol). After 100 minutes of stirring, the solution is treated with1N hydrochloric acid (1.2 mL, 1.2 mmol) and the organic solvents areevaporated in vacuo. Water (5 mL) is added and the aqueous layer isextracted with methylene chloride (3×10 mL). The combined organic phasesare dried over anhydrous magnesium sulfate, filtered and concentratedunder reduced pressure. The solid residue is triturated with ether toafford(R)-[2-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid dimethyl ester, [α]_(D) =+34.36 (c 0.84, CHCl₃).

Hydrolysis according to example 1 yields(R)-[2-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-ethylamino]-methylphosphonicacid.

(b) Similarly prepared is the corresponding (S)-enantiomer of example 1.

EXAMPLE 10

Similarly prepared according to procedures in the previous examples are:

(1)(S)-[3-(biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-prop-2-ylamino]methylphosphonicacid.

The starting material is prepared as follows:

Similarly to a method reported in J. Med. Chem., 1988, 31, 2199, to astirred solution of(S)-2-t-butoxycarbonylamino-3-(biphenyl-4-yl)-propionic acid (J. Org.Chem., 1992, 57, 379; 1 g, 2.93 mmol) in THF (10 mL) at 0° is addedN-methylmorpholine (0.35 mL, 3.18 mmol), followed by isobutylchloroformate (0.39 mL, 3.0 mmol). The suspension is stirred for 1 hour,then filtered. The precipitate is washed with dry ether (5 mL). Asaturated solution of diazomethane in ether is added at 0° untilpersistence of a yellow color. After stirring for 1 hour at 0° and 1hour at room temperature, the solution is concentrated in vacuo to yieldthe intermediate diazoketone as a beige solid (m.p. 128°-129° ). Thesolid is suspended in methanol (10 mL). A solution of silver benzoate(150 mg, 0.65 mmol) in triethylamine (3 mL) is added dropwise. The darksolution is stirred at room temperature for 30 minutes, then filteredthrough Celite. The filtrate is concentrated under reduced pressure andthe residue is redissolved in ethyl acetate (20 mL). The organic layeris washed successively with water (10 mL), saturated sodium bicarbonate(10 mL) and 1N hydrochloric acid (10 mL) before being dried overanhydrous sodium sulfate and filtered. After evaporation of the solventin vacuo, the residue is purified by flash-chromatography on silica gel,eluting with 25% ethyl acetate in hexane.(S)-3-t-Butoxycarbonylamino-4-(biphenyl-4-yl)-butyric acid methyl esteris obtained as a solid, m.p. 86°-87°.

Hydrolysis with methanolic 1N sodium hydroxide yields the carboxyclicacid which is then converted to the product using methodology similar tothat described in example 1.

(2)(S)-[4-(biphenyl-4-yl)-1-(1H-tetrazol-5-yl)-butyl-3-amino]-methylphosphonicacid

The starting material is prepared as follows:

Similarly to a method reported in Tetrahedron Lett., 1991, 923, to astirred solution of(S)-2-t-butoxycarbonylamino-3-(biphenyl-4-yl)-propionic acid (3 g, 8.8mmol) in dimethoxyethane (DME; 8 mL) at -15° is added N-methylmorpholine(0.975 mL, 8.8 mmol), followed by isobutyl chloroformate (1.2 mL, 9.25mmol). After 5 minutes, the precipitate is removed by filtration andwashed with DME (5 mL). The filtrate is cooled to 0° and treated at oncewith a freshly prepared clear solution of sodium borohydride (500 mg) inwater (5 mL). After the strong evolution of gas has ceased, water (100mL) is added and the product is extracted in ethyl acetate. The organiclayer is separated, dried over magnesium sulfate, decolorized withactivated charcoal, filtered and concentrated in vacuo to yield(S)-2-(t-butoxycarbonylamino)-3-(biphenyl-4-yl)-propan-1-ol as a whitesolid, m.p.: 116°, [α]_(D) =-22.90 (c 0.74, MeOH).

To a solution of oxalyl chloride (0.535 mL, 6.14 mmol) in methylenechloride (3 mL) cooled to -70° under nitrogen is added dropwisedimethylsulfoxide (0.830 mL, 10.7 mmol) in methylene chloride (3 mL).After 20 minutes of stirring at -70°, a solution of(S)-2-t-butoxycarbonylamino-3-(biphenyl-4-yl)-propan-1-ol (1 g, 3.05mmol) in methylene chloride (3 mL) is added, followed by triethylamine(2 mL, 14.3 mmol). The solution is allowed to warm up to roomtemperature over a 1 hour period then poured into brine. Methylenechloride (50 mL) and water (80 mL) are added. The organic layer isseparated, dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo. The crude aldehyde is dissolved in methylenechloride (15 mL) and treated with(carboethoxymethylene)-triphenylphosphorane (2.1 g, 6.1 mmol). Afterstirring at room temperature for 18 hours, silica gel (2 g) is added andthe solvent is evaporated under vacuum. The product is purified byflash-chromatography on silica gel, eluting with 25% ethyl acetate inhexane. (S)-4-(t-Butoxycarbonylamino)-5-(biphenyl-4-yl)-pentenoic acidethyl ester is obtained a white solid, m.p. 91°-94°.

Catalytic hydrogenation, e.g. with palladium on charcoal, and hydrolysiswith methanolic 1N sodium hydroxide yields(S)-4-(t-butoxycarbonylamino)-5-(biphenyl-4-yl)pentanoic acid, and suchis then converted to the product using methodology similar to thatdescribed in example 1.

EXAMPLE 11

Preparation of 1,000 capsules each containing 25 mg of the activeingredients, as follows:

    ______________________________________                                        (S)-[2-Biphenyl-4-yl-1-(1H-tetrazol-5-yl)-                                                                25.00 g                                           ethylamino]-methylphosphonic acid diphenyl ester                              Lactose                    192.00 g                                           Modified starch             80.00 g                                           Magnesium stearate          3.00 g                                            ______________________________________                                    

Procedure: All the powders are passed through a screen with openings of0.6 mm. The drug substance is placed in a suitable mixer and mixed firstwith the magnesium stearate, then with the lactose and starch untilhomogenous. No. 2 hard gelatin capsules are filled with 300 mg of saidmixture each, using a capsule filling machine.

Analogously capsules are prepared, containing about 10-100 mg of theother compounds disclosed and exemplified herein.

What is claimed is:
 1. A compound of formula ##STR24## or a tautomerthereof wherein R₁ is aryl or biaryl; n is zero, 1 or 2; apharmaceutically acceptable mono- or di-ester derivative thereof inwhich one or both of the acidic hydroxy groups of the phosphonofunctional group are esterified in form of a pharmaceutically acceptablemono- or di-ester; or a pharmaceutically acceptable salt thereof.
 2. Acompound of the formula ##STR25## wherein R₁ represents monocycliccarbocyclic aryl, monocyclic heterocyclic aryl, or monocycliccarbocyclic aryl which is substituted by monocyclic carbocyclic ormonocyclic heterocyclic aryl; monocyclic heterocyclic aryl within theabove definitions represents thienyl optionally substituted by loweralkyl, furanyl optionally substituted by lower alkyl, pyridyl optionallysubstituted by lower alkyl, halogen or cyano, pyrrolyl or N-loweralkylpyrrolyl; n is zero, 1 or 2; a pharmaceutically acceptable mono- ordi-ester derivative thereof in which one or both of the acidic hydroxygroups of the phosphono functional group are esterified in form of apharmaceutically acceptable mono- or di-ester; said ester being an aryl,tetrahydronaphthyl or indanyl mono- or di-ester, an α-acyloxymethylmono- or di-ester optionally substituted by lower alkyl, by C₅ -C₇-cycloalkyl, by aryl or by aryl-lower alkyl, or a lower alkyl oraryl-lower alkyl mono- or di-ester each substituted on the α-carbon bycarboxy, by esterified or amidated carboxy or by trichloromethyl; or apharmaceutically acceptable salt thereof.
 3. A compound according toclaim 2 of the formula ##STR26## and a tautomer thereof wherein R and R'represent independently hydrogen, carbocyclic aryl,6-tetrahydronaphthyl, 5-indanyl, α-(trichloromethyl, carboxyl,esterified carboxyl or amidated carboxyl) substituted-(lower alkyl oraryl-lower alkyl), acyloxymethyl optionally monosubstituted on methylcarbon by lower alkyl, by C₅ -C₇ -cycloalkyl, by aryl or by aryl-loweralkyl; R₂ represents phenyl, or phenyl substituted by lower alkyl, loweralkoxy, halogen or trifluoromethyl; or R₂ represents thienyl or furanyloptionally substituted by lower alkyl; R₃ and R₄ represent hydrogen,lower alkyl, hydroxy, lower alkoxy or halogen; n is 0, 1 or 2; or apharmaceutically acceptable salt thereof.
 4. A compound according toclaim 3 wherein R and R' independently represent hydrogen, carbocyclicaryl, α-(trichloromethyl, carboxyl, esterified carboxyl or amidatedcarboxyl) substituted-(lower alkyl or aryl-lower alkyl), or (carbocyclicaroyloxy or lower-alkanoyloxy)methyl optionally substituted on themethyl carbon by lower-alkyl, by C₅, C₆ or C₇ -cycloalkyl or bycarbocyclic aryl; R₂ represents phenyl or phenyl substituted by loweralkyl, lower alkoxy, halogen or trifluoromethyl; R₃ and R₄ representhydrogen, lower alkyl, lower alkoxy, halogen or trifluoromethyl; or apharmaceutically acceptable salt thereof.
 5. A compound according toclaim 4 wherein R and R' independently represent hydrogen, 5-indanyl,phenyl, or phenyl substituted by one, two or three substituents selectedfrom lower alkyl, halogen, lower alkoxy, lower alkanoylamino,trifluoromethyl, lower alkyl (thio, sulfinyl or sulfonyl) and loweralkoxycarbonyl.
 6. A compound according to claim 4 wherein R and R'independently represent hydrogen or α-(carboxy, lower alkoxycarbonyl,carbocyclic arylmethoxycarbonyl, aminocarbonyl or mono- or di-loweralkylaminocarbonyl) substituted-(lower alkyl or carbocyclic aryl-loweralkyl).
 7. A compound according to claim 1 of formula IIa ##STR27## andtautomers thereof wherein R and R' independently represent hydrogen,carbocyclic aryl, 5-indanyl, α-(carboxy, lower alkoxycarbonyl,carbocyclic arylmethoxycarbonyl, aminocarbonyl or mono- or di-loweralkylaminocarbonyl) substituted-(lower alkyl or carbocyclic aryl-loweralkyl), R₄ and R₄ ' independently represent hydrogen, lower alkyl, loweralkoxy, halogen or trifluoromethyl; n is zero or 1; or apharmaceutically acceptable salt thereof;
 8. A compound according toclaim 7 of formula III ##STR28## or a tautomer thereof wherein R₄ and R₄' represent hydrogen or C₁ -C₃ alkoxy; or a pharmaceutically acceptablemono- or di-ester derivative thereof in which one or both of the acidichydroxy groups of the phosphono functional group are esterified in formof a pharmaceutically acceptable mono- or di-ester; a pharmaceuticallyacceptable salt thereof.
 9. A compound according to claim 8 of formula##STR29## or a tautomer thereof wherein Ar represents phenyl or phenylsubstituted by lower alkyl, lower alkoxy, halogen, trifluoromethyl,lower alkanoylamino, lower alkyl-(thio, sulfinyl or sulfonyl) or loweralkoxycarbonyl; or Ar represents 5-indanyl; R₅ represents hydroxy, loweralkoxy, aryl-lower alkoxy or di-lower alkylamino; R₆ represents hydrogenor lower alkyl; R₄ and R₄ ' independently represent hydrogen or C₁ -C₃alkoxy; or a pharmaceutically acceptable salt thereof.
 10. A compoundaccording to claim 8 being the (S)-enantiomer.
 11. A compound accordingto claim 9 being the (S)-enantiomer.
 12. A compound according to claim 8being(S)-[2-(biphenyl)-4-yl-1-(tetrazol-5-yl)-ethylamino]-methylphosphonicacid or a pharmaceutically acceptable salt thereof; or apharmaceutically acceptable pro-drug ester thereof.
 13. A compoundaccording to claim 8 being(S)-[2-(2-methoxy-biphenyl-4-yl)-1-(tetrazol-5-yl)-ethylamino]-methylphosphonicacid or a pharmaceutically acceptable salt thereof; or apharmaceutically acceptable prodrug ester thereof.
 14. A compoundaccording to claim 8 being(S)-[2-(2'-methoxy-biphenyl-4-yl)-1-(tetrazol-5-yl)-ethylamino]-methylphosphonicacid or a pharmaceutically acceptable prodrug ester thereof.
 15. Acompound according to claim 9 being(S)-[2-biphenyl-4-yl-1-(tetrazol-5-yl)-ethylamino]-methylphosphonic aciddiphenyl ester.
 16. A compound according to claim 9 being(S)-[2-biphenyl-4-yl-1-(tetrazol-5-yl)-ethylamino]-methylphosphonic acidmonophenyl ester.
 17. A neutral endopeptidase inhibiting pharmaceuticalcomposition comprising an effective neutral endopetisdase inhibitingamount of a compound of claim 1 in combination with one or morepharmaceutically acceptable carriers.
 18. A method of treatingcardiovascular disorders in mammals which comprises administering to amammal in need thereof an effective neutral endopeptidase inhibitingamount of a compound of claim
 1. 19. A method of treating cardiovasculardisorders in mammals which comprises administering to a mammal in needthereof an effective neutral endopeptidas inhibiting amount of acompound of claim
 15. 20. A method of treating cardiovascular disordersin manmmals which comprises administering to a mammal in need thereof aneffective neutral endopeptidase inhibiting amount of a compound of claim2.